Characterization of the nitric oxide reductase from Thermus thermophilus

Nitrous oxide (N2O) is a powerful greenhouse gas implicated in climate change. The dominant source of atmospheric N2O is incomplete biological dentrification, and the enzymes responsible for the release of N2O are NO reductases. It was recently reported that ambient emissions of N2O from the Great Boiling Spring in the United States Great Basin are high, and attributed to incomplete denitrification by Thermus thermophilus and related bacterial species [Hedlund BP, et al. (2011) Geobiology 9(6)471–480]. In the present work, we have isolated and characterized the NO reductase (NOR) from T. thermophilus. The enzyme is a member of the cNOR family of enzymes and belongs to a phylogenetic clade that is distinct from previously examined cNORs. Like other characterized cNORs, the T. thermophilus cNOR consists of two subunits, NorB and NorC, and contains a one heme c, one Ca2+, a low-spin heme b, and an active site consisting of a high-spin heme b and FeB. The roles of conserved residues within the cNOR family were investigated by site-directed mutagenesis. The most important and unexpected result is that the glutamic acid ligand to FeB is not essential for function. The E211A mutant retains 68% of wild-type activity. Mutagenesis data and the pattern of conserved residues suggest that there is probably not a single pathway for proton delivery from the periplasm to the active site that is shared by all cNORs, and that there may be multiple pathways within the T. thermophilus cNOR.

[1]  S. Tannenbaum,et al.  A mechanistic analysis of nitric oxide-induced cellular toxicity. , 1997, Nitric oxide : biology and chemistry.

[2]  G. Butland,et al.  A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases. , 1999, Biochemistry.

[3]  R. Gennis,et al.  The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[4]  J. Moura,et al.  Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica. , 2011, Biochemistry.

[5]  R. Huber,et al.  Structure and mechanism of the aberrant ba3‐cytochrome c oxidase from Thermus thermophilus , 2000, The EMBO journal.

[6]  T. Tomizaki,et al.  The Whole Structure of the 13-Subunit Oxidized Cytochrome c Oxidase at 2.8 Å , 1996, Science.

[7]  P. Lachmann,et al.  Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase. , 2009, Journal of inorganic biochemistry.

[8]  H. Sugimoto,et al.  Molecular structure and function of bacterial nitric oxide reductase. , 2012, Biochimica et biophysica acta.

[9]  F. Cava,et al.  The role of the nitrate respiration element of Thermus thermophilus in the control and activity of the denitrification apparatus. , 2008, Environmental microbiology.

[10]  R. Gennis,et al.  The cytochrome ba3 oxygen reductase from Thermus thermophilus uses a single input channel for proton delivery to the active site and for proton pumping , 2009, Proceedings of the National Academy of Sciences.

[11]  K. Kataoka,et al.  Diverse NO reduction by Halomonas halodenitrificans nitric oxide reductase. , 2005, Biochemical and biophysical research communications.

[12]  D. Kastrau,et al.  Nitric oxide reductase from Pseudomonas stutzeri, a novel cytochrome bc complex. Phospholipid requirement, electron paramagnetic resonance and redox properties. , 1994, European journal of biochemistry.

[13]  J. Reimann,et al.  A pathway for protons in nitric oxide reductase from Paracoccus denitrificans. , 2007, Biochimica et biophysica acta.

[14]  D. Richardson,et al.  Defining the Proton Entry Point in the Bacterial Respiratory Nitric-oxide Reductase* , 2008, Journal of Biological Chemistry.

[15]  T. Martínez,et al.  Comparative genomics and site-directed mutagenesis support the existence of only one input channel for protons in the C-family (cbb3 oxidase) of heme-copper oxygen reductases. , 2007, Biochemistry.

[16]  Todd J Martinez,et al.  Evolutionary migration of a post-translationally modified active-site residue in the proton-pumping heme-copper oxygen reductases. , 2006, Biochemistry.

[17]  K. Imahori,et al.  Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a Nonsporulating Thermophilic Bacterium from a Japanese Thermal Spa , 1974 .

[18]  H. Nikaido,et al.  Multidrug Efflux Pump MdtBC of Escherichia coli Is Active Only as a B2C Heterotrimer , 2009, Journal of bacteriology.

[19]  H. Schulz,et al.  Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli. , 1998, Biochemical and biophysical research communications.

[20]  W. Zumft,et al.  Formation of the N-N bond from nitric oxide by a membrane-bound cytochrome bc complex of nitrate-respiring (denitrifying) Pseudomonas stutzeri , 1989, Journal of bacteriology.

[21]  Paul G Falkowski,et al.  The Evolution and Future of Earth’s Nitrogen Cycle , 2010, Science.

[22]  Yi Lu,et al.  Roles of glutamates and metal ions in a rationally designed nitric oxide reductase based on myoglobin , 2010, Proceedings of the National Academy of Sciences.

[23]  S. Ferguson,et al.  The nitric oxide reductase of Paracoccus denitrificans. , 1990, The Biochemical journal.

[24]  J. Berenguer,et al.  Lateral Transfer of the Denitrification Pathway Genes among Thermus thermophilus Strains , 2010, Applied and Environmental Microbiology.

[25]  M. Blomberg,et al.  Mechanism for N₂O generation in bacterial nitric oxide reductase: a quantum chemical study. , 2012, Biochemistry.

[26]  G. Butland,et al.  Two Conserved Glutamates in the Bacterial Nitric Oxide Reductase Are Essential for Activity but Not Assembly of the Enzyme , 2001, Journal of bacteriology.

[27]  M. Blomberg,et al.  Reduction of nitric oxide in bacterial nitric oxide reductase--a theoretical model study. , 2006, Biochimica et biophysica acta.

[28]  M. Hill,et al.  Integrity of thermus thermophilus cytochrome c552 Synthesized by escherichia coli cells expressing the host‐specific cytochrome c maturation genes, ccmABCDEFGH: Biochemical, spectral, and structural characterization of the recombinant protein , 2000, Protein science : a publication of the Protein Society.

[29]  Yuji Sugita,et al.  Molecular Dynamics Simulations Reveal Proton Transfer Pathways in Cytochrome C-Dependent Nitric Oxide Reductase , 2012, PLoS Comput. Biol..

[30]  So Iwata,et al.  Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase , 2010, Science.

[31]  R. Gennis,et al.  Functional importance of a pair of conserved glutamic acid residues and of Ca(2+) binding in the cbb(3)-type oxygen reductases from Rhodobacter sphaeroides and Vibrio cholerae. , 2012, Biochemistry.

[32]  A. Pastuszyn,et al.  Cloning and Expression in Escherichia coli of the Cytochrome c 552 Gene from Thermus thermophilus HB8 , 1998, The Journal of Biological Chemistry.

[33]  H. Michel,et al.  The Structure of cbb3 Cytochrome Oxidase Provides Insights into Proton Pumping , 2010, Science.

[34]  A. Puustinen,et al.  The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site , 2000, Nature Structural Biology.

[35]  K. Furukawa,et al.  Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp , 1986, Journal of bacteriology.

[36]  T. Martínez,et al.  Helix switching of a key active-site residue in the cytochrome cbb3 oxidases. , 2005, Biochemistry.

[37]  G. Butland,et al.  A new assay for nitric oxide reductase reveals two conserved glutamate residues form the entrance to a proton-conducting channel in the bacterial enzyme. , 2007, The Biochemical journal.

[38]  Rachel Zufferey,et al.  Overproduction of theBradyrhizobium japonicum c-Type Cytochrome Subunits of thecbb3Oxidase inEscherichia coli , 1998 .

[39]  A. Ravishankara,et al.  Nitrous Oxide (N2O): The Dominant Ozone-Depleting Substance Emitted in the 21st Century , 2009, Science.

[40]  B. Hungate,et al.  Potential role of Thermus thermophilus and T. oshimai in high rates of nitrous oxide (N2O) production in ∼80 °C hot springs in the US Great Basin , 2011, Geobiology.

[41]  T. Sakurai,et al.  Genomic DNA cloning of the region encoding nitric oxide reductase in Paracoccus halodenitrificans and a structure model relevant to cytochrome oxidase. , 1998, Biochemical and biophysical research communications.

[42]  S. Kawabata,et al.  Nitric oxide-reductase homologue that contains a copper atom and has cytochrome c-oxidase activity from an aerobic phototrophic bacterium Roseobacter denitrificans. , 2002, Journal of biochemistry.

[43]  T. Uchida,et al.  Structural characterization of a binuclear center of a Cu-containing NO reductase homologue from Roseobacter denitrificans: EPR and resonance Raman studies. , 2004, Biochimica et biophysica acta.

[44]  M. Brunori,et al.  The cytochrome cbb3 from Pseudomonas stutzeri displays nitric oxide reductase activity. , 2001, European journal of biochemistry.

[45]  Hartmut Michel,et al.  Structure at 2.8 Å resolution of cytochrome c oxidase from Paracoccus denitrificans , 1995, Nature.

[46]  Satoshi Takahashi,et al.  NO Reduction by Nitric-oxide Reductase from Denitrifying Bacterium Pseudomonas aeruginosa , 2004, Journal of Biological Chemistry.

[47]  Yuji Sugita,et al.  Crystal structure of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus , 2012, Nature Structural &Molecular Biology.

[48]  T. Hollocher,et al.  Purification and some characteristics of nitric oxide reductase-containing vesicles from Paracoccus denitrificans. , 1989, The Journal of biological chemistry.